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Dengue viruses (DENV) are enveloped single-stranded positive-sense RNA viruses transmitted by Aedes spp. mosquitoes. There are four genetically distinct serotypes designated DENV-1 through DENV-4, each further subdivided into distinct genotypes. The dengue scientific community has long contended that infection with one serotype confers lifelong protection against subsequent infection with the same serotype, irrespective of virus genotype. However this hypothesis is under increased scrutiny and the role of DENV genotypic variation in protection from repeated infection is less certain. As dengue vaccine trials move increasingly into field-testing, there is an urgent need to develop tools to better define the role of genotypic variation in DENV infection and immunity. To better understand genotypic variation in DENV-3 neutralization and protection, we designed and constructed a panel of isogenic, recombinant DENV-3 infectious clones, each expressing an envelope glycoprotein from a different DENV-3 genotype; Philippines 1982 (genotype I), Thailand 1995 (genotype II), Sri Lanka 1989 and Cuba 2002 (genotype III) and Puerto Rico 1977 (genotype IV). We used the panel to explore how natural envelope variation influences DENV-polyclonal serum interactions. When the recombinant viruses were tested in neutralization assays using immune sera from primary DENV infections, neutralization titers varied by as much as ∼19-fold, depending on the expressed envelope glycoprotein. The observed variability in neutralization titers suggests that relatively few residue changes in the E glycoprotein may have significant effects on DENV specific humoral immunity and influence antibody mediated protection or disease enhancement in the setting of both natural infection and vaccination. These genotypic differences are also likely to be important in temporal and spatial microevolution of DENV-3 in the background of heterotypic neutralization. The recombinant and synthetic tools described here are valuable for testing hypotheses on genetic determinants of DENV-3 immunopathogenesis. Author Summary: Infectious virus clones are valuable tools for studying how changes in viral genetic codes affect viral biology. Dengue virus is the most important mosquito-borne virus worldwide, yet dengue virus infectious clones have historically been challenging to make and manipulate, making it very difficult to study the variety of genetic changes observed in dengue viruses. Here we describe the construction of a panel of five dengue virus serotype 3 (DENV-3) clones using a novel strategy not previously employed in dengue research. This strategy uses genetic fragments and synthesized genes to introduce genetic changes while minimally affecting the virus. Each of the five recombinant clones was designed to express genetically distinct DENV-3 envelope proteins derived from strains circulating in different regions of the world. We used the recombinant viruses, coupled with DENV-3 sera from geographically defined human cases, to study the impact of E variation on neutralization outcomes. Our data demonstrate that the recombinant viruses varied significantly in their neutralization outcomes, depending on sera. While it has long been presumed that infection, and vaccination, with one serotype confers lifelong protection against all variants of that serotype, our results indicate that this assumption requires a more rigorous assessment by the DENV community. [ABSTRACT FROM AUTHOR]